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Biochimica et Biophysica Acta, 383 (1975) 427--434 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98266

SIMULTANEOUS RIBOSOMAL RESISTANCE TO TRICHODERMIN AND ANISOMYCIN IN SA CCHA R 0/14 YCES C E R E VISIA E MUTANTS

ANTONIO JIMENEZ, LUCAS SANCHEZ and DAVID VAZQUEZ Inslituto de Biologia Celular, Velazquez 144, Madrid 6 (Spain) {Received October 18th, 1974)

Summary

A spontaneous mutant of Saccharomyces cerevisiae resistant to tricho- dermin has been isolated. It displays cross resistance both in vivo and in vitro to a number of sesquiterpene antibiotics (fusarenon X, trichothecin and verrucarin A) and to the chemically unrelated antibiotic anisomycin.

The mutat ion conferring resistance to anisomycin and trichodermin is ex- pressed in the 60-S subunit of the yeast 80-S ribosome. Mutant ribosomes bind [14C] trichodermin much less efficiently than wild type ribosomes, suggesting that resistance may be due, at least in part, to this property. However, both types of ribosomes bind [ ~H]anisomycin equally. These results suggest that anisomycin and trichodermin have different binding sites on the 60-S subunit of eukaryotic ribosomes, even though previous results have shown that both antibiotics bind to mutually exclusive sites.

Introduct ion

A number of compounds of the sesquiterpene group of antibiotics have been shown to inhibit protein synthesis at the level of the eukaryotic 80-S ribosomes [1--3]. Trichodermin, trichothecin and fusarenon X and also the chemically unrelated antibiotic anisomycin block in model systems the pep- tidyltransferase center of the 60-S subunit [2,4,5]. [14C]Trichodermin and [ 3 H] anisomycin bind to the 60-S subunit of yeast ribosomes and compete with each other for their binding to the peptidyltransferase center [4,5]. Moreover, fusarenon X and trichothecin inhibit the binding of [14C]trichodermin and [ 3H] anisomycin, while verrucarin A only induces partial inhibition of binding [4,5]. A mutant resistant to trichodermin has been isolated that displays cross resistance in vitro to other sesquiterpene antibiotics and in vivo to anisomycin [6,7]. Taken together, these findings indicate that all these antibiotics have intimately related binding sites on the 60-S yeast ribosome subunit, and similar modes of action.

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In the present communicat ion we report the isolation of another yeast mutant resistant to trichodermin that is cross-resistant both in vivo and in vitro to several sesquiterpene antibiotics and anisomycin. The results show that the mutat ion conferring resistance to anisomycin and trichodermin is expressed in the 60-S subunit. The binding of [14C]trichodermin and [~H]anisomycin to sensitive and resistant ribosomes shows that resistance to trichodermin is proba- bly caused by a decreased affinity of the altered ribosome. Resistance to aniso- mycin is most likely due to a ribosomal conformational and/or structural change which renders the mutant ribosome less susceptible to the action of the drug, since [ ~H]anisomycin binds with equal affinity to both types of ribo- somes. Anisomycin and trichodermin have different, although mutually exclu- sive sites of binding on the yeast ribosome.

Materials and Methods

Yeast strains. Wild type haploid Saccharomyces cerevisiae strain Y166 (a, his4, trps, MA1) has been described elsewhere [8]. TRI mutant strain was obtained spontaneously by plating Y166 cells on YEPD medium (1% yeast extract, 2% peptone, 2% glucose and 2% agar) plates containing 5 pg/ml tricho- dermin. Strain 373/19 (a, ade2) was a gift from Dr B. Littlewood.

Cell growth and labelling conditions. Yeast cells were grown in yeast nitrogen base-dehydrated medium (Difco) at 30°C. Growth was followed by measuring the absorbance at 660 nm (0.15 A6s0 nm unit per 107 cells per ml). The uptake of [3H]leucine in intact cells was determined as described else- where [9].

Genetic studies. A diploid strain was obtained by crossing TR, and 373/19 cells for 24 h followed by selection on minimal plates. Genetic studies by random spore analysis and tetrad dissection were carried out by standard techniques [10].

Ribosomes and supernatant fractions. High salt-washed ribosomes were obtained from frozen cells stored at --27°C as described by Battaner and Vazquez [11]. Polysomes were obtained from spheroplasts basically as de- scribed [8,12]. The cells were harvested by centrifugation in a GS3 Sorvall rotor for 5 min at 10 000 X g, washed with one volume of sterile water, sedi- merited again by centrifugation and finally resuspended in 1/10 their volume of 1 M sorbitol. Glusulase (ENDO Laboratories) was added to a final concentra- tion of 0.6% and the suspension was incubated for 90 min at 30°C with gentle shaking in order to obtain intact spheroplasts. These spheroplasts were then incubated for 3 h at 30°C in one volume of a medium [8,12] enriched with yeast nitrogen base plus glucose and 0.4 M MgSO 4 for the recovery of protein synthesis activity by the spheroplast. Cycloheximide (10 pg/ml) was added at the end of the incubation period. The spheroplasts were then harvested by centrifugation in a GS3 Sorval rotor at 12 000 × g for 20 min and the pellet was resuspended in 1/60 volume of 20 mM Tris • HC1 buffer, pH 7.4, contain- ing 30 mM MgCI2 and 100 mM NaC1. Lysis was carried out with two successive incubations of 5 min at 0°C in the presence of sodium deoxycholate (0.20%) and Brij 35 (0.25%). The lysed extract was centrifuged in a SS34 Sorval rotor at 10 000 X g for 10 min. The supernatant of this centrifugation was layered

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on 4.5 ml of 60% sucrose in 50 mM Tris • HCI buffer, pH 7 .4 ,100 mM KC1, 30 mM MgC12 and centrifuged for 11 h at 45 000 rev./min in the 65 ro tor of the Spinco L-2 ultracentrifuge. Pellets were resuspended in the above buffer and kept in liquid N2 until used. These polysomes can be thawed and frozen at least six times wi thout loss of activity. The supernatant fraction was a 30--70% (NH4)2SO4 precipitate dialysed versus 20 mM Tris • HC1 buffer, pH 7.4, 1 mM dithiothreitol .

The zonal centrifugation technique developed by Van der Zeijst et al. [13] was used to prepare the 60-S and 40-S r ibosome subunits, but t rea tment with I mM NaN3 was performed in intact cells rather than in spheroplasts [121.

Cell-free protein synthesis. Poly(U)-directed polyphenylalanine synthesis using high salt-washed ribosomes or reconst i tuted 80-S ribosomes from 60-S and 40-S subunits and endogenous mRNA-programmed polypept ide synthesis on yeast polysomes were carried out as described [8 ,12] . Trichloroacetic acid precipitates were collected in glass-fiber filters (Whatman GF/C), washed three times with 5% trichloroacetic acid, dried and radioactivity estimated in a scin- tillation spectrometer [4] .

Binding of radioactively labelled antibiotics to ribosomes. [ ' 4C] Tricho- dermin (15.4 Ci/mol) and [ 3H]anisomycin (285 Ci /mol)binding to ribosomes was performed by the ultracentrifugation technique [14] . [ ' 4C] Trichodermin and [ 3H] anisomycin were prepared by Dr M. Barbacid as previously described [5 ,15] .

Antibiotics. Sources of the non-radioactive antibiotics used in this work were as previously indicated [2,5] .

Results

The effect of antibiotics on the growth of yeast cells Trichodermin and anisomycin affect the peptidyltransferase center of the

r ibosome [4,5] . Both antibiotics appear to have intimately related binding sites on the yeast 60-S r ibosome subunit. Consequently, our first aim was to check the possibility of cross resistance to anisomycin in our trichodermin-resistant strain. The growth of Y166 and TR~ strains in the presence and absence of antibiotics is shown in Fig. 1. It can be seen that TR ~ cells are fully resistant to 10 -5 M tr ichodermin and also partially resistant to 4 " 10 -4 M anisomycin. In addition TR 1 cells were resistant to 3 • 10 -s M verrucarin A and 10 -4 M tricho- thecin. Both TR 1 and Y166 cells were resistant to 5 • 10 -3 M fusarenon X most probably due to a permeabili ty barrier [16] .

Genetic studies To ascertain whether the resistance to t r ichodermin and anisomycin was

the effect of a single mutat ion, a diploid strain was constructed following a cross between TR 1 and 373/19, and induced to sporulate as described in Mate- rials and Methods. Random spore analysis showed that all the spores resistant to anisomycin were also resistant to t r ichodermin and that an approximate ratio of resistant to sensitive haploids of 1 to 1 was obtained. Moreover, six tetrads were dissected by micromanipulat ion and segregated two sensitive and

430

c

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j l~ , - - " , y .Y.

~<?i, o o~

o 1 2 3 4 5 6

TIME (HOURSI

o. o 7 j I . . . . . . . .

7 2 3 /. 5 6

TIME (HOURS}

Fig. 1. T h e e f f e c t o f a n i s o m y c i n a n d t r i c h o d e r m i n o n y e a s t ce l l s g r o w t h . Y 1 6 6 a n d T R 1 ce l l s w e r e g r o w n

in m i n i m a l m e d i a a t 3 f f C in s i d e - t u b e c o n i c a l f l a sk s . A t t h e t i m e i n d i c a t e d b y t h e a r r o w s e i t h e r t r i c h o -

d e r m i n o r a n i s o m y c i n w a s a d d e d a n d i n c u b a t i o n w a s c o n t i n u e d . ( A ) Y 1 6 6 c o n t r o l ( e ) ; Y 1 6 6 + 1 0 -5 M

t r i c h o d e r m i n (A): T R 1 c o n t r o l a n d T R | + 1 0 -5 M t r i c h o d e r m i n ( ' - ) . (B) Y 1 6 6 c o n t r o l ( o ) ; Y 1 6 6 +

4 - 1 0 - 4 M a n i s o m y c i n ( A ) ; T R 1 c o n t r o l ( ~ ' ) ; T R 1 + 4 " 1 0 -4 M a n i s o m y c i n (A).

two resistant spores. In all cases, resistance to t r ichodermin and to anisomycin were always associated. The effect of anisomycin was tested on poly(U)- directed polyphenylalanine synthesis by cell-free extracts from the offsprings of each spore. The results obtained showed that spores which were resistant to t r ichodermin and anisomycin in vivo had an identical phenotype in vitro. More- over, a second spontaneous, independent ly isolated trichodermin-resistant yeast strain was also found to be resistant in vivo to anisomycin [7] . It is therefore concluded that , most probably, a single muta t ion confers resistance to tricho- dermin and anisomycin.

The effect o f antibiotics on in vitro polypept ide synthesis Fig. 2 shows the effect of several antibiotics of the tr ichodermin group

and anisomycin, at different concentrat ions, on endogenous RNA-programmed polypept ide synthesis and poly(U)-directed polyphenylalanine synthesis. Poly- somes from Y166 strain are sensitive to tr ichodermin, t r ichothecin, fusarenon X and anisomycin and polysomes from TR1 cells are cross-resistant to these four antibiotics {Figs 2A--2C and 2E). Fig. 2 also shows that the same pattern of effects was obtained when polyphenylalanine synthesis was studied. These findings agree with previous results for another trichodermin-resistant mutant [6 ,7] . Trichodermin has been previously shown to have a very small inhibitory effect on poly(U)-directed polyphenylalanine synthesis, although other anti- biotics of the tr ichothecin group are good inhibitors of polyphenylalanine syn- thesis [2 ,6] . This is one of the reasons we have also included in our studies a very active cell-free system which uses yeast polysomes. Verrucarin A was found to be a rather poor inhibitor of polypept ide synthesis on polysomes even at high drug concentrat ions (Fig. 1D) which is in agreement with the previous finding that this antibiotic induces polysomal run off and hence affects a step in polypept ide initiation [17] . Fusarenon X, which has been shown to induce polysome degradation in HeLa cells in vivo [16 ,18] , is a good inhibitor of

4 3 1

"~ 20! ~ "~" 20

TRICHODERMIN CONC (mM) TRICHOTHECIN CONC (raM)

100,B0

~ ' ' ' - ~ ~-~ i ̧ 2

0 ~' , '~ 3 1'0_ 2 - - 10.1

FUSARENON X CONC (raM)

]

<"--.i

10-5 .10 - ~ -- i~-3(

VERRUCARIN A CONC(mM)

'~ E

o '%3 10-~ ,0-' ANISOMYCIN CONC (raM)

F ig . 2. Yeas t r i b o s o m e s e n d o g e n o u s m R N A - d i r e c t e d p o l y p e p t i d e and p o l y ( U ) - d i r e e t e d p o l y p h e n y l a l a n i n e syn thes is . T h e r e a c t i o n m i x t u r e s e o n t a i n e d t he f o l l o w i n g c o m p o n e n t s i n a f i n a l v o l u m e o f 50 /~]: 50 m M Tris • HCI buf fe r , pH 7.4, 12.5 m M MgCI2, S0 m M KCI, 4 mM crea t ine p h o s p h a t e , 2 pg c rea t ine phospho- kinase, 125 pg yeas t t R N A , 1 r a m ATP; 0 .05 m M GTP; 0 .0 7 5 mM [ 1 4 C ] p h e n y l a l a n i n e (New England Nuclear , l S 0 Ci /mol ) ; s u p e r n a t a n t e n z y m e s ( a b o u t 2 m g / m l p ro te in ) and e i ther 0.8 A 2 6 0 n m un i t of high sa l t -washed r i bosomes plus 15 #g of p o l y ( U ) or 0.6 A 2 6 0 n m un i t of p o l y s o m e s w h e n requ i red . Incuba - t ions (45 rain at 30°C) were fo l lowed by p rec ip i t a t i on wi th 1 ml of 10% t r i ch lo roace t i c acid. Po lypheny l - a lanine synthes is (-- ); p o l y p e p t i d e synthes is on p o l y s o m e s ( . . . . . ). Y166 ex t rac t s (e) ; TR 1 ex t r ac t s (¢'D. I n c o r p o r a t i o n in the un inh ib i t ed con t ro l s averaged 19.99 or 4 9 2 .5 (Y166 ex t rac ts ) and 16 .44 or 501 .68 (TR 1 ex t rac t s ) pmols of [ 14C] pheny la l an ine for e n d o g e n o u s m R N A - or po ly (U) -d i r ec t ed poly- pep t idc synthesis , respect ive ly . S t anda rd dev ia t ion was ± 12%.

polypeptide elongation in our systems (Fig. 2C). The results with verrucarin A in our cell-free polysome system fully agree with those obtained by others in vivo [17] .

Effect of anisomycin and fusarenon X on reassociated ribosomes The results presented in Table I, show the data obtained on polyphenyl-

4 3 2

T A B L E I

T H E E F F E C T O F A N I S O M Y C I N A N D F U S A R E N O N X O N P O L Y ( U ) - D I R E C T E D P O L Y P H E N Y L A L A -

N I N E S Y N T H E S I S B Y R I B O S O M E S R E C O N S T I T U T E D F R O M R I B O S O M A L S U B U N I T S

I n c u b a t i o n s w e r e c a r r i e d o u t as d e s c r i b e d in t h e l e g e n d o f F ig . 2 b u t y e a s t r i b o s o m e s w e r e r e p l a c e d b y

0 . 8 A2~,0 n m u n i t o f 60 -S s u b u n i t s a n d 0 . 4 A 2 6 0 n m u n i t o f 40 -S s u b u n i t s . I n c o r p o r a t i o n t o o k p l a c e f o r 1 0 r a i n a t 30ZC. V a l u e s in b r a c k e t s r e p r e s e n t t h e p e r c e n t a g e o f c o n t r o l s .

S u b u n i t s [ 14C ] P h e n y l a l a n i n e i n c o r p o r a t e d p e r r e a c t i o n m i x t u r e ( p m o l )

6 0 S 4 0 S C o n t r o l + A n i s o m y c i n w i t h o u t + F u s a r e n o n X

a n t i b i o t i c 2 • 10 -6 M 2 • 10 -5 M 2 . 10 -4 M

s e n s i t i v e s e n s i t i v e 1 5 1 . 6 ( 1 0 0 ) 9 2 . 0 ( 6 1 ) 3 3 . 6 ( 2 2 ) 1 8 . 5 ( 1 2 ) r e s i s t a n t r e s i s t a n t 1 1 6 . 2 ( 1 0 0 ) 1 1 2 . 9 ( 9 7 ) 8 0 . 6 ( 6 9 ) 1 0 1 . 5 ( 8 7 ) r e s i s t a n t s e n s i t i v e 1 3 4 . 0 ( 1 0 0 ) 1 2 5 . 2 ( 9 3 ) 8 6 . 0 ( 6 4 ) 1 0 3 . 8 ( 7 8 )

s e n s i t i v e r e s i s t a n t 1 3 8 . 7 ( 1 0 0 ) 9 4 . 4 ( 6 8 ) 3 6 . 6 ( 2 6 ) 2 2 . 3 ( 1 6 )

alanine synthesis by reassociated ribosomes. Only the 60-S subunit is affected by the mutation. Since trichodermin is a poor inhibitor of polyphenylalanine synthesis, we have used fusarenon X in these experiments. This antibiotic cross- reacts with trichodermin and effectively blocks polyphenytalanine synthesis.

[14 C| Trichodermin and [3 H] anisomycin binding to ribosomes Barbacid and Vazquez [4,5] have shown that trichodermin and aniso-

mycin bind to yeast ribosomes at mutually exclusive sites. Also, both anti- biotics are inhibitors of the peptidyltransferase center of the 60-S subunit [2 ,5] . These results suggest a proximity or overlapping between their binding

T A B L E II

B I N D I N G O F [ 1 4 C ] T R I C H O D E R M I N A N D [ 3 H ] A N I S O M Y C I N T O Y E A S T S E N S I T I V E A N D R E S I S - T A N T R I B O S O M E S

R e a c t i o n m i x t u r e s ( 1 0 0 /21), m a d e up in 0 . 6 m l n i t r o c e l l u l o s e t u b e s c o n t a i n e d t h e f o l l o w i n g c o m p o n e n t s : 50 m M T r i s . HC1 b u f f e r , p H . 7,4, 1 2 . 5 m M M g C I 2 , 80 m M KC1 a n d 3 . 7 " 10 - 3 m M r i b o s o m e s a n d e i t h e r [ 1 4 C ] t r i c h o d e r m i n o r [ 3 H ] a n i s o m y e i n . T h e t u b e s w e r e c e n t r i f u g e d f o r 2 . 5 h a t 3 8 0 0 0 r e v . / m i n in t h e 4 0

r o t o r o f t h e S p i n e o u l t r a c e n t r i f u g e . 30-#1 s a m p l e s w e r e t a k e n b e f o r e a n d a f t e r c e n t r i f u g a t i o n f o r d e t e r - m i n a t i o n o f r a d i o a c t i v i t y . F i g u r e s in b r a c k e t s r e p r e s e n t t h e p e r c e n t a g e o f b i n d i n g o f t o t a l a n t i b i o t i c a d d e d .

R i b o s o m e s nM c o n e e n - n m o l b o u n d t r a t i o n r a d i o a c t i v e a n t i b i o t i c | 14C] - [ 3 H ] -

a d d e d T r i e h o d e r m i n A n i s o m y c i n

Y 1 6 6 1 0 0 4 .9 ( 6 1 ) 5 .1 ( 5 1 )

T R 1 1 0 0 0 . 4 ( 5 ) 5 . 0 ( 5 0 ) Y 1 6 6 2 0 0 1 1 . 4 ( 5 7 ) 8 . 4 ( 4 3 ) T R 1 2 0 0 2 .0 ( 1 0 ) 1 1 . 3 ( 5 7 ) Y 1 6 6 5 0 0 2 0 . 7 ( 4 1 ) 19 .1 ( 3 8 ) T R 1 5 0 0 7 .4 ( 1 5 ) 2 0 . 0 ( 4 0 )

433

sites. To study the effect of the alteration in mutan t ribosomes on their interac- tion with anisomycin and trichodermin we have carried out binding experi- ments using both radioactive antibiotics. High salt-washed ribosomes from strain TR1 have a reduced capacity to bind [ 14C] trichodermin at several drug concentrations as compared with wild type ribosomes (Table II). Thus resis- tance to trichodermin in TR, mutan t cells is most probably due to a lower affinity of the ribosomes for the drug. Surprisingly, ribosomes from Y166 and TR~ strains bound equal amounts of [ 3 HI anisomycin at several concentrations of the antibiotic (Table II). Thus it appears that resistance to anisomycin in TR, cells is due to an alteration of the mutant ribosomes that renders the ribosome less sensitive to the antibiotic although it does not significantly modi- fy its affinity for anisomycin. Moreover, these results strongly suggest that anisomycin and trichodermin do not bind precisely to the same ribosomal sites.

Discussion

The results presented in this contribution show that mutat ion to tricho- dermin resistance in yeast bears a concomitant resistance to anisomycin. Ge- netic studies strongly suggest that a single mutat ion is responsible for the resistance to these two drug, which have quite different chemical structures but rather similar modes of action [1,4,5]. Moreover, the in vitro results shown in Figs 2A--2E give evidence that resistance to trichodermin also induces resis- tance to other sesquiterpene antibiotics (trichothecin, fusarenon X and verru- carin A).

It has been demonstrated that anisomycin and trichodermin bind to mutu- ally exclusive sites on ribosomes and also that both antibiotics are inhibitors of peptide-bond formation [1,4,5]. These findings suggest overlapping binding sites and similar modes of action for both antibiotics. In agreement with these results we have found that mutat ion to resistance towards trichodermin also induces resistance to anisomycin. The results of binding studies with radio- actively labelled [J 4 C] trichodermin and [ 3 H] anisomycin to sensitive and resis- tant ribosomes show that resistance to trichodermin is due to a lower binding affinity of mutant ribosomes for the antibiotic (Table II). On the other hand, there are no significant differences in the amounts of [ ~H] anisomycin bound by sensitive and resistant ribosomes (Table II). These results suggest that, as a consequence of the ribosomal mutat ion leading to trichodermin resistance, the peptidyltransferase center is altered in such a manner that it is less affected by the interaction of anisomycin with the 60-S subunit. A similar situation has been described in ribosomes, from an erythromycin-resistant strain of Escheri- chia coli; ribosomal protein L22 is altered in this mutant [19].

Acknowledgements

This work has been supported by grants from "Fondo Nacional para el Desarrollo de la Investigacidn Cientffica" and Lilly Indiana of Spain. We are grateful to Drs Vidal and Santamarfa for their help in the dissection of the asci and to Miss A. Martfn for technical assistance.

434

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